Up to now protein profiles have been acquired in mass spectrometry using ionization by matrix-assisted laser desorption (MALDI) with high detection sensitivity in linear time-of-flight mass spectrometers, but these display very poor mass resolution and a very poor reproducibility of the mass values. A recently devised method permits very easy, and largely automated, identification of microbes, especially bacteria, by mass spectrometry. In this method, small quantities of microbes are first taken from a colony grown overnight on a nutrient medium and transferred to a mass spectrometric sample support plate. The microbes are then sprinkled with a solution of a customary matrix substance for ionization using matrix-assisted laser desorption (MALDI). This solution penetrates into the microbial cells and destroys them during the crystallization of the matrix material in the subsequent drying process. Proteins and peptides, and possibly other analyte substances of the cell, are integrated into the matrix crystals. The dry sample with the matrix crystals is then bombarded with pulsed flashes of laser light in the vacuum of a time-of-flight mass spectrometer, creating pulses of ions of the analyte substances, which can then accelerated and be measured in the time-of-flight mass spectrometer. The mass spectrum is the profile of the measured ion current values of these peptide ions, protein ions and other analyte ions of the microbial material. This profile is very characteristic of the microbe species concerned. It is even possible to distinguish between substrains of microbes because their composition of proteins, defined by the genes in a one-to-one translation without much modifications, is very characteristic. Even small changes in the genes generate proteins of slightly different masses detectable by mass spectrometry. The identification appears to be extremely reliable, as far as current analyses have shown. It does not require individual identification of the proteins involved.
In a similar way, protein profiles are acquired in the search for so-called “biomarkers”. Biomarkers are indicators of stress situations in organisms, whether diseases, chemical and pharmacological stress, ageing, physical stress caused by heat or impact, or stress with other causes. Biomarkers are represented as up or down regulated proteins caused by the stress. Biomarkers are read out from mass spectra of the protein profile samples obtained from body fluids or tissue homogenates. The samples can contain either all proteins or only extracted sub-quantities of proteins. As the protein profiles themselves usually display some fluctuations in signal intensity, in most cases a statistical evaluation is required, for which mass spectra of cohorts of “normal samples” or “healthy samples” (samples from healthy individuals) are compared with mass spectra from other cohorts of “stress samples” or “disease samples” (samples from diseased individuals). The biomarkers are obtained by statistical evaluation of the ion signals in the mass spectra. These biomarkers can be individual proteins which are over-expressed or under-expressed to a statistically significant degree, or they can be characteristic intensity patterns of relatively large numbers of proteins, i.e. the biomarkers can only be expressed as mathematical or logical expressions containing intensities of a variety of ion signals.
For these applications, mass spectra are acquired at present in linear time-of-flight mass spectrometers because of their particularly high detection sensitivity, even though the mass resolution and mass accuracy of the spectra from time-of-flight mass spectrometers with reflectors are incomparably superior. In reflector mode, however, only about a twentieth of the ion signals appear, and the detection sensitivity is inferior by several orders of magnitude. The inadequate quality of the mass spectra in time-of-flight mass spectrometers operated in linear mode is partially due to the formation process for ions by matrix-assisted laser desorption in vacuum (vacuum-MALDI). Vacuum-MALDI delivers ions of widely differing initial velocity distributions and differing mean initial energies
The processes during ionization of the analyte substances in the laser-induced vaporization cloud are not easily reproducible; they depend greatly on structural inhomogeneities of the microcrystalline sample after it has been prepared. Furthermore, the uneven thickness of the sample after its preparation causes the formation of ions at differing initial potentials, with the result that they pass through varying potential differences, and therefore absorb slightly different energies, according to the location where they were formed. These effects influencing the flight times of the ions can be partly eliminated, for example by means of delayed acceleration, but they cannot all be corrected simultaneously. On account of the change in the flight times of the ions from spectrum to spectrum, the mass scales of the spectra are distorted because these are calculated from the flight times using the calibration curve, which is determined once.
The acquisition of mass spectra with time-of-flight mass spectrometers generally requires a very large number of individual spectra, which are usually added together, measuring value by measuring value, to form sum spectra. The ions for the individual spectra are generated by a laser shot each. This procedure of generating sum spectra is made necessary by the low measuring dynamics in the individual spectrum. At least about 50, and in some cases even 1,000 or more, individual spectra are acquired; in general, a sum spectrum consists of several hundred individual spectra. By adding up spectra with different mass values for identical substances the resolution of the mass spectrum is greatly deteriorated.
In the linear operating mode of a time-of-flight mass spectrometer, it is possible to detect not only the stable ions, but also the fragment ions from so-called “metastable” decompositions of the ions, and even neutral particles that are formed from the ion decompositions along the way. All these fragment ions and neutral particles which have resulted from a single parent ion species have the same velocity as the parent ions and therefore reach the ion detector at the same time. In the applications described above, this gives a ten-fold to hundred-fold detection sensitivity. For these applications, the energy of the desorbing and ionizing laser is raised, thereby increasing the ion yield, but also their instability. This increased detection sensitivity is of such decisive importance for many applications that the disadvantages of linear operation of time-of-flight mass spectrometers described above are more or less accepted.
These decompositions of the ions always add to the inferiority of the mass resolution. When an ion decomposes, a small excess of internal energy is always released, which is transferred to the two fragments of the ion as kinetic energy. Depending on the direction of the decomposition in relation to the direction of flight, the particles may be slightly accelerated or slightly decelerated. This results in a broader distribution of the flight times of particles that have the same parent ion mass, and that in turn reduces the mass resolution. This reduction in resolution is thus inseparably connected with the increase in detection sensitivity, and cannot, in principle, be removed. The mass resolutions are only around R=400 to R=1,000.
The non-reproducibility of the mass scale described above means that no easily comparable mass spectra are obtained. It is difficult, for example, to create a good reference spectrum library for identifying microbes on the basis of their protein profiles. Spectra of the same microbes from different sample preparations do not match exactly, but display apparently different mass values for what are actually identical proteins. Deviations of up to one percent of the mass value have been observed.
The non-reproducibility of the mass scales of linear TOF mass spectra is particularly bothering if promising biomarker patterns have been found, validated by thousands of samples, and now should be used for diagnostic assays for diseases. There is a large field of future applications of mass spectrometry in medicine. In medicine, however, very strong rules apply to the reproducibility of measuring results. The application of linear time-of-flight mass spectrometers without safe methods to correct for distorted mass scales or without better reproducibility will presumably not permitted by validating organizations.
In the applications described above, mass spectra up to high mass ranges of, for example, 20,000 Daltons are measured. For reasons of low mass resolution, as mentioned, the isotope groups, which consist of several ion signals that differ by one Dalton respectively, cannot be resolved in major parts of the mass spectrum. Therefore, only the envelopes of the isotope groups are measured, a fact that makes mass determination and a corresponding calibration even more difficult. Furthermore, protein profile spectra in particular are very rich in ion signals, with many ion signal overlaps, which greatly impedes the comparison of patterns. The protein profile spectra usually contain the ion signals of several hundred different proteins.
Time-of-flight mass spectrometers with reflectors have a very much better mass resolving power, in particular because no fragment masses contribute to the mass spectrum. Unfortunately, this means that protein profile spectra, whether for searching for biomarkers or for identifying microbes, cannot be acquired in reflector mode. The mass spectra in reflector mode only show around a twentieth of the ion signals, albeit with far better mass resolution, but the wealth of mass spectra obtained with the linear mode, and the associated capacity for biomarker search or microbe identifications, is lost. Furthermore, the detection sensitivity is drastically reduced, although mass-resolved mass spectra generally display higher detection sensitivity on account of the better signal-to-noise ratio.
There have been made trials to replace vacuum MALDI by atmospheric pressure electrospray ionization in combination with high resolution mass spectrometers for biomarker searching. But the generation of large amounts of doubly, triply and even quadruply charged ions creates much too complex spectra; and the richness of the spectra with ion signals does no longer permit to distinguish between singly and multiply charged ion species. MALDI, in contrast, has the advantage of yielding singly charged ions only. The use of electrospray ionization in combination with a separation of the substances of the complex mixtures in biomaterial by chromatography or capillary electrophoresis does in principle work, but requires much longer measuring times per sample (hours instead of seconds), and increases the amount of data to be handled by factors of thousands.
In most raw biomaterial samples like blood, plasma, homogenized tissue, spinal liquid and many others, the complexity of proteins is even too high for linear mode time-of-flight mass spectra generated by vacuum-MALDI. Raw biomaterial samples usually contain thousands of proteins. There are, however, solutions to this problem. The complexity of the biomaterial can be reduced by several methods, e.g. by broad-band extraction of subsets of proteins with magnetic beads that have been activated at their surfaces to bind groups of proteins by different types of affinity. Magnetic beads are available with different degrees of hydrophobicity, with cation and anion exchange phases, with different immobilized metals, or even with proteins acting as ligands, e.g. antibodies. Each of these types of magnetic beads can reduce the extract to several hundred types of proteins, thereby reducing the complexity considerably.
Beside vacuum-MALDI, there have been also different approaches to generate ions by MALDI at atmospheric pressure (AP-MALDI). The ionization can take place, just as in vacuum-MALDI, by means of protonation by matrix substance ions which occur in the plasma of the laser evaporation cloud. Such “normal” AP-MALDI at atmospheric pressure with protonation by the matrix substance is proposed in the patents U.S. Pat. No. 5,965,884 (V. V. Laiko and A. L. Burlingame) and EP 0 964 427 A2 (J. Bai et al.). This ionization at atmospheric pressure seems to have a higher yield of analyte ions because the analyte molecules and the matrix substance ions are kept together for longer time by the inert gas than is the case in the vacuum, and therefore display a better protonation yield. On the other hand, the introduction of the ions into the vacuum causes the vast majority of the ions to get lost, with the result that this method has a lower overall detection sensitivity than a method with production of vacuum-MALDI ions in vacuum. The yield of analyte ions generated in vacuum-MALDI amounts to about a ten-thousandth of the evaporated analyte molecules; at atmospheric pressure, the yield of evaporated analyte molecules of AP-MALDI is assumed to amount to roughly a thousandth.
In contrast to this commercially available AP-MALDI, the patent U.S. Pat. No. 5,663,561 (J. Franzen and C. Köster) proposes to avoid the background created by the usual matrix substances by the use of decomposable matrix substances and to greatly increase the yield of analyte ions at atmospheric pressure by post-ionization of the analyte molecules. As only around a thousandth of the analyte molecules is ionized by the usual AP-MALDI process, there is a large potential for increasing the detection sensitivity here. The post-ionization can be carried out by photo ionization using a UV lamp, for example, or by chemical ionization, which produces a particularly high yield of analyte and is therefore preferred.
Chemical ionization (CI) usually starts with an electron source generating a very large number of electrons, which in turn generate many primary ions of an inert gas which can be nitrogen, for example. Usually suitable substances to form reactant ions, sometimes called “mediators”, such as low amounts of water, methane, butane, or even xylene, are added to the inert gas, The inert gas ions then react immediately (within some ten nanoseconds) with the water molecules, forming OH3+, O2H5+ and higher protonated water clusters. Within microseconds, these water cluster ions form protonated methane clusters or reactant ions from the other mediator substances. The protonated reactant ions are then available for the protonation of the analyte molecules. By this rather complicated reaction chain reactant ions are formed which react with the analyte ions in such a manner that virtually no fragment ions occur during protonation of the analyte molecules. Chemical ionization (CI) is therefore termed “soft”. Only singly charged non-fragmented ions are generated by this process.
In APCI (atmospheric pressure chemical ionization), corona discharges at the tip of corona discharge needles under high voltage or 63Ni beta radiation emitters can produce a large amounts of reactant ions which in turn may ionize the majority of the analyte molecules. The 63Ni beta radiation emitter, in the form of a ring-shaped foil with a diameter of approximately one centimeter and a width of two millimeters can be easily mounted around the sample gas volume as known from ion mobility spectrometry.
In another type of atmospheric pressure ionization (API), the reactant ions can, however, already have been added to the inert gas when the latter is fed to the sample as a purging flow, as also described in the patent U.S. Pat. No. 5,663,561, referred to above. The sample is then desorbed by laser shots into the flowing inert gas. But in this case, different flows of gases have to mix, the desorption plume on one hand, and the inert gas with the reactant ions on the other hand. The expanding desorption plume first pushes aside the inert gas without mixing. Because mixing at atmospheric pressure takes time in the order of many milliseconds even for small volumes, this procedure is much less efficient than expected.
Electron beams can also be generated by photon bombardment of suitable surfaces with an adequately low work function. The surface can be bombarded with UV radiation from a UV lamp or a UV laser, either continuously or in pulses. A withdrawal potential of several kilovolts draws the electrons away from the surface and accelerates them to a level where they are able to generate large quantities of inert gas ions. These ions can build up the chain of reactant ions.